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Chromatography, Reverse-Phase: A chromatography technique in which the stationary phase is composed of a non-polar substance with a polar mobile phase, in contrast to normal-phase chromatography in which the stationary phase is a polar substance with a non-polar mobile phase.

High-Performance Liquid Chromatography (HPLC)

JoVE 10156

Source: Dr. Paul Bower - Purdue University

High-performance liquid chromatography (HPLC) is an important analytical method commonly used to separate and quantify components of liquid samples. In this technique, a solution (first phase) is pumped through a column that contains a packing of small porous particles with a second phase bound to the surface. The different solubilities of the sample components in the two phases cause the components to move through the column with different average velocities, thus creating a separation of these components. The pumped solution is called the mobile phase, while the phase in the column is called the stationary phase. There are several modes of liquid chromatography, depending upon the type of stationary and/or mobile phase employed. This experiment uses reversed-phase chromatography, where the stationary phase is non-polar, and the mobile phase is polar. The stationary phase to be employed is C18 hydrocarbon groups bonded to 3-µm silica particles, while the mobile phase is an aqueous buffer with a polar organic modifier (acetonitrile) added to vary its eluting strength. In this form, the silica can be used for samples that are water-soluble, providing a broad range of applications. In this experiment, the mixtures of three components frequently found


 Analytical Chemistry

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification

1St. Jude Proteomics Facility, St. Jude Children's Research Hospital, 2Departments of Structural Biology and Developmental Neurobiology, St. Jude Children's Research Hospital, 3Heilongjiang University of Chinese Medicine

Video Coming Soon

JoVE 56474


 JoVE In-Press

Sample Extraction and Simultaneous Chromatographic Quantitation of Doxorubicin and Mitomycin C Following Drug Combination Delivery in Nanoparticles to Tumor-bearing Mice

1Department of Pharmaceutical Sciences, University of Toronto, 2Departments of Medical Biophysics and Radiation Oncology, University of Toronto, Ontario Cancer Institute, University Health Network

Video Coming Soon

JoVE 56159


 JoVE In-Press

Construction of Models for Nondestructive Prediction of Ingredient Contents in Blueberries by Near-infrared Spectroscopy Based on HPLC Measurements

1United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, 2Faculty of Agriculture, Tokyo University of Agriculture and Technology, 3Institute of Agriculture, Tokyo University of Agriculture and Technology

JoVE 53981


 Chemistry

Sample Preparation for Endopeptidomic Analysis in Human Cerebrospinal Fluid

1Inst. of Neuroscience and Physiology, Dept. of Psychiatry and Neurochemistry, Sahlgrenska Academy at University of Gothenburg, 2Clinical Neurochemistry Laboratory, Sahlgrenska University Hospital, 3Department of Molecular Neuroscience, UCL Institute of Neurology

Video Coming Soon

JoVE 56244


 JoVE In-Press

Synthesis and Characterization of Charged Hydrogels for the Extended Delivery of Vancomycin

1Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Graduate School, Mayo Clinic College of Medicine, Mayo Clinic, 2Pharmacometrics Center, Duke Clinical Research Institute, 3Department of Laboratory Medicine and Pathology, Mayo Clinic, 4Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Mayo Clinic, 5Department of Physiology and Biomedical Engineering, Mayo Clinic College of Medicine, Mayo Clinic, 6Department of Orthopedic Surgery, Mayo Graduate School, Mayo Clinic College of Medicine, Mayo Clinic

Video Coming Soon

JoVE 55517


 JoVE In-Press

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

1Reference and Translation Center for Cardiac Stem Cell Therapy (RTC), Department of Cardiac Surgery, University of Rostock, 2Physikalisch-Technische Bundesanstalt, 3Department of Radiology and Neuroradiology, Ernst-Moritz-Arndt-University Greifswald, 4Electron Microscopy Center, University of Rostock

JoVE 55567


 Medicine

Chromatography-based Biomolecule Purification Methods

JoVE 5683

In biochemistry, chromatography-based purification methods are employed to isolate compounds from a complex mixture. Two such methods used commonly by biochemists are size-exclusion chromatography and affinity chromatography. In size-exclusion chromatography, a column packed with porous beads separates components of a mixture based on size. On the other hand, affinity chromatography allows for a more specific separation of biomolecules by using a column that is composed of stationary phase, which contains target-specific ligands. This video serves as an introduction to size-exclusion and affinity chromatography, as well as the concepts that govern them. A step-by-step procedure for the purification of a histidine-tagged protein by immobilized metal affinity chromatography is described. Applications for both of these chromatographic methods in biochemistry and biomedical research are also profiled. "Chromatography" refers to a wide range of methods used to isolate a component from a complex mixture, an essential step before a biomolecule's properties and activities can be determined. Each chromatographic technique has a different mechanism for separation, depending on the sample matrix and target compound. This video will focus on the principles and operation of two methods common to biochemistry: size-exclusi


 Biochemistry

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